When faced to a hypertonic environment, cells adapt to it by accumulating compatible osmolytes, which reduces intracellular inorganic salts and as a result protects the cell from the deleterious effects of the high intracellular inorganic salt concentration. Being exposed to variably high levels of osmolality due to the urinary concentrating mechanism, renal medullary cells protect themselves by accumulating myo-inositol, which is a known compatible osmolyte. This is primarily through the up-regulation of transporter protein, called sodium/myo-inositol cotransporter (SMIT). Previous studies have indicated that transcription is the primary step in the up-regulation of SMIT activity and Kwonet. al. in my lab identified five putative cis-acting sequences involved in the regulation of the SMIT gene. These are tonicity-responsive enhancers (TonEs) spread over 55 kilobase pairs in the 5'-flanking region of the SMIT gene promoter. We hypothesize that there are special structural features in the chromatin that allow all TonEs to interact with SMIT promoter. To test our hypothesis, I will prepare a 100 kb yeast artificial chromosome containing the human SMIT locus-the entire gene and 60 kb of flanking sequence containing all the enhancers. Taking advantage of homologous recombination, variations of the locus will be prepared by deleting the enhancers and altering their location. Then I will transfect MDCK cell line with this construct to assess the effects of the changes. I will also investigate whether there are other genes present between TonEs and SMIT promoter and will study their possible regulation by hypertonicity in MDCK cells transfected with human SMIT YAC construct and its variations.